Restoring functional connections within the nervous system following injury or disease represents a significant challenge in the development of effective treatments for stroke, spinal cord injury, and neurodegenerative conditions. Because the adult nervous system is relatively resistant to the functional restoration of neural circuits, t is believed that recapitulating programs used during the initial formation of these circuits holds significant promise for encouraging nerve regeneration following injury or disease. During embryonic development, nerve growth is guided by minute gradients of extracellular guidance cues. How these guidance cue signals are locally amplified within growth cones at the tip of elongating nerves remains a critical question. Evidence from a number of model systems that exhibit chemotactic growth towards extracellular signals indicates that positive feedback loops are often used to locally amplify extracellular cues and generate the cellular asymmetry required for guided cell migration. The goal of this proposal is to ascertain whether similar mechanisms are at work in neuronal growth cones, and to define the components and function of these signaling pathways. Based on work by other groups and our own, WE HYPOTHESIZE that extracellular guidance cue signals are asymmetrically amplified within the growth cone by a positive feedback loop comprised of the lipid signaling molecule phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3), the lipid kinase PI-3 kinase (PI3K), the Rho GTPase Rac, and actin dynamics. Furthermore, we propose that Ca2+ functions in a positive feedback loop with PIP3 in growth cones to promote the localized accumulation of PIP3 and Rac activity. In order to characterize these putative positive feedback loops in growth cones, we will use pharmacological and molecular approaches along with innovative biosensors in live neuronal cultures to identify the components and function of a PIP3-PI3K-Rac-actin dynamics positive feedback loop (aim 1), as well as the contribution of cytoplasmic Ca2+ signaling to the amplification of asymmetric PIP3 signals and growth cone turning (aim 2). Collectively, the work described in this proposal will provide novel insight into how local guidance cue signals are amplified and contribute to the growth cone symmetry breaking required for turning and outgrowth to proper synaptic targets. PUBLIC HEALTH RELEVANCE: The adult nervous system is relatively resistant to significant functional regeneration following stroke, injury, or neurodegenerative disease. It is widely believed that therapeutic interventions which can more closely recapitulate developmental processes in the adult nervous system have the greatest potential for restoring damaged or diseased neural circuits. The goal of this proposal is to understand how extracellular guidance cue signals are locally amplified within developing or regenerating nerves to ensure the proper wiring of the nervous system.